Custom made rabbit polyclonal anti gfp antibodies

Carboxyl latex beads (860 nm in diameter, Life Technologies) were coated with custom-made rabbit polyclonal anti-GFP antibodies (Covance). 200 μL of beads were resuspended three times in activation buffer (10 mM MES pH 6.0, 100 mM NaCl) after spinning down at 8,000 rpm for 3 min. 1 mg of EDC and 1 mg of S-NHS were dissolved in 1 mL and 2 mL DMF, respectively and 20 μL of dissolved EDC and 40 μL of dissolved S-NHS were added to beads. The beads were sonicated for 3 min and nutated at low speed until visible clumps disappeared. The beads were rinsed in PBS (phosphate-buffered saline, pH 7.4) buffer in 800 μL total volume and reacted with 200 μL of 0.4 mg ml⁻¹ of antibody. After shaking the mixture for 30 min, the beads were passivated by adding 10 mg ml⁻¹ BSA for 2 h, washed in PBS three times and stored in PBS supplemented with 0.1% sodium azide and 0.5 mg ml⁻¹ BSA at 4 °C.